Antibody(-based) libraries have been extensively used in the past for the selection and identification of proteins specifically binding to one or more target compounds of interest.
The applicability of the various types of antibody libraries varies. For instance, immune libraries are characterized by a high percentage of high affinity antibodies directed to a limited amount of target antigens (immunogens), as in such libraries in vivo antibody affinity maturation has taken place in immunized animals or human patients. Therefore, immune libraries with a relatively small complexity are capable of generating high affinity binders for specific antigens. However, usually, for each antigen or patient group studied, a separate immune library has to be constructed. In addition, for obvious ethical reasons, active immunization of humans is not possible, and accordingly the generation of human antibodies to most targets is not feasible via this approach.
On the other hand, in naïve and synthetic antibody(-based) libraries, the antibody repertoire present did not evolve through antigen driven antibody maturation. Consequently, the complexity of these libraries has to be very high to ensure a sufficient statistical chance that some of the antibodies in the libraries are capable of binding to any given target with reasonable or high affinity.
Antibodies are fairly large proteins and sensitive to treatments like heating, freeze-thawing proteolytic cleavage and ammonium sulphate precipitation. Also the size of antibodies limits their modes of administration.
Accordingly, in view of the disadvantages of the antibody scaffolds, there remains a need for alternative and improved protein libraries (and methods for the production thereof), which reliably yield good-quality proteins having a high affinity and activity towards a variety of target proteins of interest.
WO2010/066740 and EP 2 161 278 describe Alphabody structures as single-chain triple-stranded alpha-helical coiled coil scaffolds. However, up to now it has not been disclosed or suggested how the Alphabody scaffolds can be manipulated to obtain Alphabody libraries which yield a high number of binding partners capable of specifically binding to target molecules of interest with sufficient affinity.